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1 CBS Fungal Biodiversity Centre, Uppsalalaan 8, NL-3584 CT Utrecht, The
Netherlands
2 Korean Agricultural Culture Collection, NIAB, Suwon, 441-707,
Korea
3 Microbial Genomics and Bioprocessing Research Unit, National Center for
Agricultural Utilization Research, 1815 N. University Street, Peoria, IL
61604, U.S.A.
4 BioCentrum-DTU, Building 221, Technical University of Denmark, DK-2800
Kgs. Lyngby, Denmark
5 University of Szeged, Faculty of Science and Informatics, Department of
Microbiology, P.O. Box 533, H-6701 Szeged, Hungary
*
Correspondence: Robert A. Samson,
r.samson{at}cbs.knaw.nl
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Abstract |
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Taxonomic novelties: Neosartorya australensis, N. ferenczii, N. papuaensis, N. warcupii.
Keywords Aspergillus section Fumigati / extrolite profiles / Neosartorya / phylogenetics / polyphasic taxonomy
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INTRODUCTION |
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Aspergillus fumigatus Fresenius is an ubiquitous filamentous fungus in the environment, and also an important human pathogen (Raper & Fennell 1965). Several Neosartorya species have been described as causal agents of human diseases including invasive aspergillosis, osteomyelitis, endocarditis and mycotic keratitis (Coriglione et al. 1990; Summerbell et al. 1992; Padhye et al. 1994; Lonial et al. 1997; Jarv et al. 2004; Balajee et al. 2005, 2006). All of the Neosartorya species produce heat-resistant ascospores that are frequently encountered in different food products (Gomez et al. 1994; Samson 1989; Tournas 1994). The several mycotoxins produced by these species may cause serious health hazard (Fujimoto et al. 1993; Frisvad & Samson 1990; Larsen et al. 2007). Some species also have valuable properties for mankind; e.g. N. fischeri strains produce fiscalins which effectively inhibit the binding of substance P to the human neurokinin receptor (Wong et al. 1993), while A. fumigatus strains produce pyripyropenes, potent inhibitors of acyl-CoA:cholesterol acyltransferase (Tomoda et al. 1994), the immunosuppressant restrictocins (Müllbacher & Eichner 1984), ribotoxins (Lin et al. 1995) and fumagillin that has amebicidal activity (McCowen et al. 1951). Neosartorya spinosa can be used for the complete enzymatic recovery of ferulic acid from corn residues (Shin et al. 2006).
Here we present an overview of the species belonging to Aspergillus section Fumigati based on analysis of macro- and micromorphology, extrolite profiles and β-tubulin, calmodulin, ITS and actin gene sequences of the isolates. We also describe four new homothallic Neosartorya species found in soil samples in Australia and Papua New Guinea using this polyphasic approach and list synonymies.
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MATERIALS AND METHODS |
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Morphology and physiology
The strains (Table 1) were
grown for 7 d as 3-point inoculations on Czapek agar, Czapek yeast autolysate
agar (CYA), oat meal agar (OA) and malt extract agar (MEA) plates at 25
°C, and on CYA at 37 °C. For Neosartorya species Hay infusion
agar and SNA agar have also been used for inducing the anamorphs (medium
compositions in Samson et al.
2004). In some species e.g. N. tatenoi the anamorph could
only be produced when growing the cultures at 30 or 37 °C on MEA + 40 %
sucrose.
Analysis for extrolites
Extrolites were analysed using the HPLC-diode array detection method of
Frisvad & Thrane (1987,
1993) as modified by
Smedsgaard (1997). Extrolites
were analyzed from cultures grown on CYA, OA and YES agar using three agar
plugs (Smedsgaard 1997).
Isolation and analysis of nucleic acids
Isolates used for the molecular studies were grown on 2 mL of malt peptone
broth [10 % (v/v) malt extract (Brix 10) and 0.1 % (w/v) bacto peptone
(Difco)], in 15 mL tubes. The cultures were incubated at 25 °C for 7 d.
DNA was extracted from the cells using the MasterpureTM yeast DNA
purification kit (Epicentre Biotechnol.) following the instructions of the
manufacturer. Fragments containing the ITS region were amplified using primers
ITS1 and ITS4 as described (White et
al. 1990). Amplification of partial β-tubulin gene was
performed using the primers Bt2a and Bt2b and methods of Glass & Donaldson
(1995). Amplifications of the
partial calmodulin and actin genes were as described (Hong et al.
2005,
2007). Sequencing reactions
were performed with the Big Dye Terminator Cycle Sequencing Ready Reaction Kit
and carried out for both strands. All the sequencing reactions were purified
by gel filtration through Sephadex G-50 (Amersham Pharmacia Biotech,
Piscataway, NJ) equilibrated in double-distilled water and analyzed on the ABI
PRISM 310 Genetic Analyzer (Applied Biosystems). The complementary sequences
were corrected with the MT Navigator software (Applied Biosystems). Unique
ITS, β-tubulin, actin and calmodulin sequences were deposited in GenBank
(http://www.ncbi.nlm.nih.gov)
with accession numbers DQ534140
[GenBank]
, DQ534141
[GenBank]
and EU20279-EU220287.
Data analysis
Sequence alignments were performed using CLUSTAL-X
(Thompson et al.
1997) and improved manually. The neighbour-joining (NJ) method was
used for the phylogenetic analysis. For NJ analysis, the data were first
analysed using the Tamura-Nei distance calculation with gamma-distributed
substitution rates (Tamura & Nei
1993), which were then used to construct the NJ tree with MEGA v.
3.1 (Kumar et al.
2004). A bootstrap analysis was performed with 1 000 replications
to determine the support for each clade,.
PAUP v. 4.0 b10 software was used for parsimony analysis (Swofford 2002). Alignment gaps were treated as a fifth character state and all characters were unordered and of equal weight. Maximum parsimony analysis was performed for all data sets using the heuristic search option with random addition order (100 reps) and tree bisection-reconnection (TBR) branch-swapping algorithm. Branches of zero length were collapsed and all multiple, equally parsimonious trees were saved. The robustness of the trees obtained was evaluated by 1 000 bootstrap replications (Hillis & Bull 1993). Sequences from an A. clavatus isolate were used as outgroups in these experiments.
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RESULTS AND DISCUSSION |
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Possible synonymies of some species described previously have also been examined during this study. Based on multilocus sequence analyses Hong et al. (2007) discussed the synonymy of N. botucatensis, N. paulistensis and N. takaki with N. spinosa (Raper & Fennell) Kozak. (1972). N. spinosa and the synonyms have roughly circular arrangements of projections on the ascospore convex walls. N. spinosa produces echinulate ascospores with spines ranging from < 0.5 µm up to 5(-7) µm long with verruculose and small triangular projections or sometimes with circularly arranged projections.
N. otanii Takada, Y. Horie & Abliz (2001) was described on the basis of its rapid growth on Czapek and malt extract agars, lenticular ascospores with two widely separated equatorial crests, tuberculate or lobate-reticulate convex surface, and globose to broadly ellipsoidal conidia with a microtuberculate wall. The morphology of N. otanii resembles N. fennelliae, although Takada et al. (2001) reported small differences of the ascospore ornamentation, which was not confirmed in our SEM studies. The β-tubulin gene sequences of N. otanii (GenBank accession numbers AB201363 [GenBank] and AB201362 [GenBank] ) were identical with N. fennelliae (KACC 42228) (Fig. 5A). These N. fennelliae isolates produced ascospores after mating with the N. fennelliae type strains (data not shown). N. otanii is probably synonymous with N. fennelliae, but mating experiments with N. fennelliae and N. otanii are needed for its confirmation. These experiments could not be carried out because the ex type cultures of N. otanii were not available.
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Neosartorya primulina Udagawa, Toyaz. & Tsub. (1993) was characterised by its restricted growth on Czapek agar, chalky-buff ascomata, and lenticular ascospores with a very irregular ornamentation composed of several narrow crests and verrucose hemispheres. The ascospore ornamentation and anamorph morphology resembles those of N. quadricincta. Furthermore, the ex type culture (CBS 253.94) of N. primulina showed nearly identical sequences with strains of N. quadricincta for β-tubulin, calmodulin and actin genes (Fig. 5B). N. primulina is reduced to synonymy with N. quadricincta.
Neosartorya delicata H.Z. Kong (1997) was described based on its ellipsoid or nearly clavate vesicles, and ascospores with conspicuous spines, joining one spine to another by fairly prominent ridges and reticulate ornamentation, the ridges spreading to the equatorial crests. This species has identical ascospore morphology with N. tatenoi (Fig. 36), and both taxa were clustered into a clade in three gene trees (99.6 % in β-tubulin, 98.5 % in calmodulin and 97.3 % in actin gene sequences) (Fig. 5B). Therefore, we consider N. delicata as a synonym of N. tatenoi.
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Morphology and extrolite production
The atypical N. glabra isolate NRRL 4179
(Raper & Fennell 1965)
produced asperfuran, aszonalenin, fumigaclavine, viridicatumtoxin, and
fumigatins, extrolites common in N. fennelliae, but none of the
extrolites produced by N. glabra. However, in contrast with the
heterothallic N. fennelliae, this isolate is homothallic. It is
closely related to N. denticulata based on phylogenetic analysis of
sequence data, although their ascospore ornamentations are strikingly
different (Figs. 21,
23). Ascospore ornamentation
of NRRL 4179 is similar to that of the heterothallic N. fennelliae
(Fig. 22) with equatorial
crests much narrower, while N. denticulata has denticulate ascospores
without equatorial crests. Isolate NRRL 4179 exhibited 72 % nuclear DNA
relatedness to N. fennelliae and only 60 % relatedness to N.
glabra isolates (Peterson
1992). This isolate also yielded different mtDNA and
SmaI-digested repetitive DNA patterns from those of all the other
Neosartorya strains examined
(Rinyu et al. 2000).
Hybridisation experiments were also carried out with Neurospora
crassa mating type genes (the A idiomorph with about 6 kb
flanking sequences, or the a idiomorph flanked by about 2 kb
genomic DNA on either side) to the EcoRI digested DNA of several
teleomorphic and asexual Aspergillus strains. Hybridisation to a 1.9
kb band was observed for both mating-type strains of N. fennelliae
and isolate NRRL 4179 (Rinyu et
al. 2000). Based on these observations, isolate NRRL 4179
seems to be closely related to N. fennelliae strains. These results
are in agreement with those found using carbon source utilisation tests and
isoenzyme analysis of these strains (Varga
et al. 1997).
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CBS 112.55 was isolated from garden soil in Adelaide, Australia, and produced compounds similar to wortmannin and aszonalenin and some unique metabolites, while CBS 841.96 was isolated from Podocarpus bark in Papua New Guinea, and produced a compound related to wortmannins and some unique compounds the structures of which have not yet been elucidated (Table 2). The ascospore ornamentations of these isolates were microtuberculate, similarly to those of N. glabra and N. galapagensis. However, both isolates produced cream-coloured colonies on CYA in contrast with N. glabra which produces greyish green colonies. In phylogenetic analysis they were unrelated to any other Neosartorya species, justifying their treatment as new species. We propose four new homothallic and monotypic Neosartorya species; N. ferenczii (NRRL 4179), N. warcupii (NRRL 35723), N. australensis (CBS 112.55) and N. papuensis (CBS 841.96).
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Identification
Traditionally the identification of members of section Fumigati
were done using the colony patterns and the morphology of the conidiogenous
structures, conidia, ascomata and ascsopores. Ascospore ornamentation has been
studied by Scanning electron microscopy, but our studies have shown that
different species have similar ascospore shape and surface structure. Several
species such A. fumigatus, A. novofumigatus, fumigatiaffinis, A.
fumisynnematus and A. lentulus show strong morphological
resemblance and in the lightmicroscope these species can be difficult to be
separated. The anamorphs of Neosartorya udagawae and N.
fennelliae also show a similar morphology. Therefore we recommend that
for a correct species identification, sequence analysis should be carried out.
Our experience with sequencing the calmodin and β-tubulin gen revealed
good species delimitation and recognition. All sequences of the ex type
cultures of section Fumigati are available from specialised databases
and also from GenBank.
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List of accepted species belonging to Aspergillus section Fumigati |
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Strict anamorphic species:
Aspergillus brevipes Smith
Aspergillus duricaulis Raper & Fennell
Aspergillus fumigatiaffinis Hong, Frisvad & Samson
Aspergillus fumigatus Fresenius
Aspergillus fumisynnematus Horie, Miyaji, Nishimura, Taguchi & Udagawa
Aspergillus lentulus Balajee & Marr
Aspergillus novofumigatus Hong, Frisvad & Samson
Aspergillus turcosus Hong, Frisvad & Samson
Aspergillus unilateralis Thrower
A. brevipes var. unilateralis (Thrower)
Kozakiewicz Aspergillus viridinutans Ducker & Thrower
Teleomorph species:
Neosartorya assulata Hong, Frisvad & Samson [anamorph: A.
assulatus Hong, Frisvad & Samson]
Neosartorya aurata (Warcup) Malloch & Cain [anamorph: A. igneus Kozakiewicz]
Neosartorya aureola (Fennell & Raper) Malloch & Cain [anamorph: A. aureoluteus Samson & Gams]
Neosartorya australensis Samson, Hong & Varga, sp. nov.
Neosartorya coreana Hong, Frisvad & Samson [anamorph: A. coreanus Hong, Frisvad & Samson]
Neosartorya denticulata Samson, Hong & Frisvad [anamorph: A. denticulatus Samson, Hong & Frisvad]
Neosartorya fennelliae Kwon-Chung & Kim [anamorph: A. fennelliae Kwon-Chung & Kim]
Neosartorya ferenczii Varga & Samson, spec. nov.
Neosartorya fischeri (Wehmer) Malloch & Cain [anamorph: A. fischeranus Kozakiewicz]
Neosartorya galapagensis Frisvad, Hong & Samson [anamorph: A. galapagensis Frisvad, Hong & Samson]
Neosartorya glabra (Fennell & Raper) Kozakiewicz [anamorph: A. neoglaber Kozakiewicz]
Neosartorya hiratsukae Udagawa, Tsubouchi & Horie [anamorph: A. hiratsukae Udagawa, Tsubouchi & Horie]
Neosartorya laciniosa Hong, Frisvad & Samson [anamorph: A. laciniosus Hong, Frisvad & Samson]
Neosartorya multiplicata Yaguchi, Someya & Udagawa [anamorph: A. muliplicatus Yaguchi, Someya & Udagawa]
Neosartorya papuensis Samson, Hong & Varga, sp. nov.
Neosartorya pseudofischeri Peterson [anamorph: A. thermomutatus (Paden) Peterson]
Neosartorya quadricincta (Yuill) Malloch & Cain [anamorph: A. quadricingens Kozakiewicz]
Aspergillus fischeri var. spinosus
Raper & Fennell 1965
(basionym) Neosartorya spathulata Takada & Udagawa [anamorph: A. spathulatus Takada & Udagawa]
Neosartorya stramenia (Novak & Raper) Malloch & Cain [anamorph: A. paleaceus Samson & Gams]
Neosartorya tatenoi Horie, Miyaji, Yokoyama, Udagawa & Campos-Takagi [anamorph: A. tatenoi Horie, Miyaji, Yokoyama, Udagawa & Campos-Takagi]
Neosartorya udagawae Horie, Miyaji & Nishimura [anamorph: A. udagawae Horie, Miyaji & Nishimura]
Neosartorya warcupii Peterson, Varga & Samson, sp. nov.
Doubtful species:
Neosartorya sublevispora Someya, Yaguchi & Udagawa [anamorph:
A. sublevisporus Someya, Yaguchi & Udagawa]
Neosartorya indohii Horie [anamorph: A. indohii Horie]
Neosartorya tsurutae Horie [anamorph: A. tsurutae Horie]
Neosartorya nishimurae Takada, Horie & Abliz [anamorph: A. nishimurae Takada, Horie & Abliz]
Aspergillus brevipes Smith, Trans. Br. mycol. Soc. 35: 241. 1952. Fig. 6.
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Other no. of the type: ATCC 16899; CBS 118.53; IFO 5821; IMI 16034; IMI 51494; NRRL 2439; WB 4772 = IBT 22571; WB 4078 = IBT 22572
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Description |
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Colony colour: purple red
Conidiation: abundant
Reverse colour (CZA): dull yellow turning to reddish brown
Colony texture: velutinous
Conidial head: short columnar
Stipe: 15-50 (-100) µm, occasionally septate, heavy walled
Vesicle diam, shape: 10-18 µm, pear shaped
Conidium size, shape, surface texture: 2.8-3.5 µm, globose, spinulose
Cultures examined: CBS 467.91; WB 4772; WB 4078; CBS 118.523 = IBT 3051, all from the same original source
Diagnostic features: short heavy walled stipes, finely spinulose conidia, purple red colony colour, coloured vesicles and phialides and dark blue conidia; characterised by its vesicles borne at an angle to the stipe, as in A. viridinutans and A. duricaulis
similar species: A. duricaulis
Distribution: Australia
Ecology and habitats: soil
Extrolites: Roquefortine C, cf. meleagrin, red metabolite (not structure elucidated)
Pathogenicity: not reported
Note: previous reports on viriditoxin production of A. brevipes (Weisleder & Lillehoj 1971; Cole & Cox 1981) were based on studies of a mixed culture of A. brevipes and A. viridinutans (Peterson SW, pers. comm.)
Aspergillus duricaulis Raper & Fennell, The genus Aspergillus, 249. 1965. Fig. 7.
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Other no. of the type: ATCC 16900; IMI 172282; JCM 01735; IBT 23177; NRRL 4021; VKM F-3572; WB 4021
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Description |
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Colony colour: lily green to slate olive
Conidiation: heavy in central areas
Reverse colour (CZA): colourless to pinkish drab
Colony texture: velutinous
Conidial head: loosely columnar
Stipe: 5-50 x 3.5-5.5 µm, smooth thick walled
Vesicle diam, shape: 7-14 µm, flask shaped
Conidium size, shape, surface texture: (2.8-)3-3.3(-3.3) µm, globose, echinulate
Cultures examined: IMI 172282 = IBT 23177; CBS 481.65
Diagnostic features: echinulate conidia and weakly coloured reverse on CYA distinguish it from other anamorphic species
Similar species: A. brevipes
Distribution: Argentina
Ecology and habitats: soil
Extrolites: pseurotin A, fumagillin (found here), asperpentyn (Muhlenfeld & Achenbach 1988), duricaulic acid and asperdurin (Achenbach et al. 1985a), phthalides and chromanols (Achenbach et al. 1982a, 1985b), cyclopaldic acid and 3-O-methylcyclopolic acid (Brillinger et al. 1978; Achenbach et al. 1982b)
Pathogenicity: not reported
Aspergillus fumigatiaffinis Hong, Frisvad & Samson, Mycologia 97: 1326. 2005. Fig. 8.
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Other no. of the type: KACC 41148; IBT 12703
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Description |
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Colony colour: white, with center dull green
Conidiation: limited
Reverse colour (CZA): yellowish to greyish orange
Colony texture: floccose
Conidial head: short columnar
Stipe: 6-8 µm in diam.
Vesicle diam, shape: 18-24 µm, globose-subglobose
Conidium size, shape, surface texture: 2-3 µm, globose-subglobose, smooth
Diagnostic features: has comparatively small (sub)globose vesicles (16-24 µm); able to grow at 10 °C, and unable to grow at 50 °C
Similar species: A. fumigatus, A. lentulus, A. novofumigatus, A. fumigatiaffinis
Distribution: U.S.A., Spain
Ecology and habitats: kangaroo rat, soil, human
Extrolites: auranthine, cycloechinuline, fumigaclavines, helvolic acid, neosartorin, palitantin, pyripyropenes A, E, O & S, tryptoquivaline, tryptoquivalone
Pathogenicity: pathogenic to humans (Alcazar-Fuoli et al. 2007)
Note: exhibits high MICs to amphotericin B and several triazoles (Alcazar-Fuoli et al. 2007)
Aspergillus fumigatus Fresenius, Beitr. Mykol. 81: 18. 1863. Fig. 9.
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Type: IMI 016152, from chicken lung, Connecticut, U.S.A.
Other no. of the type: Thom 118; QM 1981; WB 163; CBS 133.61; NRRL 163; ATCC 1022; LSHB Ac71; NCTC 982; KACC 41143
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Description |
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Colour: greyish turquoise or dark turquoise to dark green to dull green
Conidiation: abundant, rarely less abundant
Reverse colour (CYA): creamy, yellow to orange
Colony texture: velutinous, st. floccose (define the abreviation st.)
Conidial head: columnar
Stipe: 50-350 x 3.5-10 µm
Vesicle diam, shape: 10-26 µm, pyriform to subclavate, sometimes subglobose, but rarely globose
Conidia length, shape, surface texture: 2-3.5(-6) µm, globose to ellipsoidal, smooth to finely rough
Cultures examined: ATCC 32722, AF71, AF 293, AF294, CBS 112389, CBS 487.65, CBS 133.61, CBS 545.65, CBS 457.75, CBS 542.75, CBS 113.26, CBS 110.46, CBS 120.53, CBS 132,54, CBS 123.59, CBS 158.71, CBS 180.76, CBS 143.89, CBS 148.89, CBS 488.90, CBS 287.95, CBS 100076, CBS 109032, CBS 386.75, CBS 286.95, CEA10, IMI 376380, NRRL 1979
Diagnostic features: Rapid growing velutinous colonies, abundant and fast conidiation, thick stipe (ca. 6-10 um), large pyriform to semi-clavate vesicle is representative morphological features of the species. However, the characteristics are various according to strains, and some stains have exceptional characteristics. The species grows at 50 °C, no growth at 10 °C.
Similar species: A. fumigatiaffinis, A. fumisynnematus, A. lentulus, A. novofumigatus, A. viridinutans.
Distribution: Worldwide distribution, cosmopolitan fungus (Pringle et al. 2005)
Ecology and habitats: soil, human
Extrolites: fumagillin, fumitoxins, fumigaclavines A & C, fumitremorgins, fumiquinazolines, gliotoxin, helvolic acid, pseurotins, pyripyropens, methyl-sulochrin, trypacidin, verruculogen
Pathogenicity: pathogenic to humans (Raper & Fennell 1965; Marr et al. 2002)
Note: no growth at 10 °C, growth at 50 °C; some isolates carry dsRNA mycoviruses (Anderson et al. 1996)
Aspergillus fumisynnematus Horie, Miyaji, Nishimura, Taguchi et Udagawa, Trans. Mycol. Soc. Japan: 34: 3-7. 1993. Fig. 10.
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Description |
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TOP
Abstract
INTRODUCTION
I = 1 µm.
